1. Field of the Invention
The present invention relates to analytical test devices which are useful for the determination of an analyte in a liquid test medium. In particular, the present invention relates to multizone test devices incorporated with two or more interactive or incompatible reagents, particularly immunoassay reagents, which provide a detectable signal upon contact with analyte from the liquid test medium.
2. Description of the Prior Art
One of the problems encountered in the manufacture of test devices comprising one or more reagent zones or layers incorporated with assay reagents is the premature interaction or migration of such reagents, either during the manufacturing process or at a time prior to application of a liquid test sample to such device. Typically, such premature interaction or migration occurs where the casting of such zones or layers involves, for example, materials such as paper, gelatin or agarose which have been hydrated in order to incorporate the various assay reagents therein, or to facilitate the formation of a multilayer test device. In particular, such premature interaction or migration of reagents occurs where a single zone or layer containing interactive assay reagents is cast in a hydrated state, as well as where two or more of such zones or layers are simultaneously cast, or where subsequent hydrated zones or layers are cast over a previously dried or gelled zone or layer. Accordingly, the hydrated states of the zones or layers permits the assay reagents to freely diffuse within or migrate between such zones or layers and prematurely interact therein during the casting and/or drying process, thereby affecting the performance of the device, and in some cases rendering the device essentially inoperative as a result of such interaction.
It is therefore desirable to immobilize, segregate or otherwise prevent the premature migration and subsequent interaction of assay reagents within the various zones or layers of an analytical test element. Variations of such analytical test elements are known in the art and described by, for example, U.S. Pat. Nos. 4,333,733 and 4,356,149. The continuous release of a reagent in an analytical element to reduce assay interference is described by U.S. Pat. No. 4,333,733 wherein the analytical element comprises a reaction zone, to which a liquid test sample is applied, and a reagent zone incorporated with a chromogenic indicator. The reagent zone is impermeable to analyte and protein interferants whereby upon application of the test sample, the liquid portion thereof permeates the reagent zone causing the indicator to be released into the reaction zone. Multilayer chemical analytical materials for the detection of urea are described by U.S. Pat. No. 4,356,149 wherein a reagent layer comprising a hydrophilic binder is incorporated with hydrophobic particles containing an assay reagent therein. The particles are water-impermeable and permeable only to gaseous reaction products, e.g., ammonia, wherein the assay reagent directly, or indirectly, reacts with the gaseous reaction product to produce a change in color.
It is also known in the art to provide multizone, or multilayer, test devices which have various immobilized reagents incorporated therein. Such immobilized reagents are generally employed in such elements or test devices to inherently separate bound and free species formed by, for example, an antigen-antibody reaction.
For example, such multilayer immunoassay analytical elements are described by European Patent Publication No. 97,952 and German Publication No. DE-OS 3329728 where an immobilized form of a binding partner, such as an immobilized antibody to an antigen, and an antigen labeled with a detectable substance are incorporated therein. Upon the application of a liquid test medium to such device, antigen from the test medium competes with labeled antigen incorporated into the device for binding to the immobilized antibody. Separation of the bound species from the free species occurs upon migration of the free species of the labeled antigen away from the immobilized zone.
Similarly, European Patent Publication Nos. 51,183 and 66,648 disclose such devices where the determination or antigen or antibody in a liquid test medium is dependent upon the competitive binding of the antigen (or antibody) with a labeled form of the antigen (or antibody) for an immobilized form of a binding partner thereof, such as immobilized antibody (or antigen).
Another of such devices is described in U.S. Pat. No. 4,446,232 which is based on the principle of competition between bound and free species of analyte for a fixed number of recognition sites on an enzyme-labeled antibody. The determination of analyte in a test sample depends upon the binding of the analyte to enzyme-labeled antibodies in one zone of the device and which then pass into another zone of the device where the enzyme activity of the enzyme-linked antibodies bound to analyte is detected. One of the zones further includes bound and immobilized analyte which competes with analyte from the test sample for binding to the enzyme-labeled antibodies and which bind and immobilize any of the enzyme-labeled antibodies which do not become bound to analyte from the test sample.
Analytical elements and test devices are also known in the art which employ immobilizing agents within a zone or zones of such devices in order to localize reaction products resulting from various specific binding assay reactions within such device.
For example, European Patent Publication Nos. 51,183 and 66, 648 suggest layers for collection of the detectable reaction product comprising hydrophilic high molecular weight substances. EP No. 66,648 further suggests the incorporation of mordanting agents in the detection layer which have a strong interaction with the detectable reaction product in order to collect the detectable reaction product therein. Such mordanting agents include cationic polymers, anionic polymers and quaternary salts.
Similarly, U.S. Pat. Nos. 4,144,306 and 4,042,335 disclose multilayer analytical elements which include a registration layer incorporated with a mordant for a detectable species in order to collect the detectable species therein and thereby prevent diffusion or migration of the detectable species out of the registration layer.
A variation of such devices is disclosed by U.S. Pat. No. 4,459,358 which describes a multilayer element comprising a spreading layer, a reaction layer incorporated with a diffusible labeled antibody, and a registration layer incorporated with materials adapted to non-specifically bind, immobilize or "mordant" antibodies, such as latex particles. Upon application of a liquid test medium to the device, analyte from the test medium associates with the labeled antibody in the reaction layer and immunoprecipitates therein. Any of the labeled antibody which does not become bound to the analyte diffuses into the registration layer where it is immobilized by the mordant incorporated therein.
However, such analytical elements and test devices are directed to the permanent immobilization of reagents which do not participate in the initial assay reactions necessary for the detection of an analyte in a liquid test medium. Although the immobilization of such reagents provides means for preventing the further migration of reaction products formed within a test device, the problem of premature migration and interaction of assay reagents necessary for the formation of reaction products which can be correlated to the amount of an analyte in a liquid test medium nevertheless remains unsolved.
Accordingly, it is an object of the present invention to provide in an analytical test device assay reagents which participate in assay reactions necessary for the determination of analyte from a liquid test medium wherein the reagents are prevented from prematurely migrating and/or interacting during manufacture and prior to application of the liquid test medium to such test device.
Another object of the present invention is to provide for the essentially instantaneous release of reversibly immobilized assay reagents in an analytical test device upon application of a liquid test medium to such test device.
Further, it is an object of the present invention to permit the simultaneous incorporation of otherwise diffusible and interactive assay reagents in a test device during the manufacturing process thereof.